Thermostable blunt-end ligase and methods of use

ABSTRACT

Thermostable blunt-end ligases suitable for use in nucleic acid ligation reactions at elevated temperatures are provided. The ligases comprise fusion proteins including a DNA ligase and a DNA binding protein, e.g., a T4 DNA ligase with an N-terminal p50 fusion. The fusion proteins may include peptide linkers, peptide mimetics, terminal additions, tag peptides, D-amino acids, sugars, non-amino acid organic moieties, and polymers. The ligases are suitable for use in ligation reactions, including uniform-temperature ligation reactions, performed at about 60° C. or higher, e.g., at about 75° C. The ligases are suitable for use in nucleic acid amplification schemes with temperature cycling, e.g., temperature cycles to about 60° C. or higher, or temperature cycles from about 94° C. to about 60° C. Such nucleic acid amplification schemes may include one, two, three, or more temperature cycles. Methods of using the ligases, and articles of manufacture comprising the ligases are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to, and the benefit under 35 U.S.C.§§120 and 365(c), of international application PCT/US2014/030003, filedMar. 15, 2014, which international application claims priority to, andthe benefit under 35 U.S.C. §119(e) of, U.S. Patent Application61/802,124, filed Mar. 15, 2013, both of which applications are herebyincorporated by reference in their entireties.

BACKGROUND

A variety of methods for the amplification of nucleic acids are known.For example, polymerase chain reaction (“PCR”) (see, e.g. U.S. Pat. No.4,683,202) is a popular method for the amplification of nucleic acids.To successfully perform a PCR reaction, the reaction must be performedat multiple different temperatures. This requires hardware or othermechanisms for repeatedly changing the temperature of the PCR reaction.Another method for amplification of nucleic acids is referred to asloop-mediated isothermal amplification (“LAMP”) (see, e.g. U.S. Pat. No.6,410,278). LAMP reactions may be performed isothermally, but typicallyinvolve the use of four different primers which recognize a total of sixdistinct sequences on the target nucleic acid.

Some amplification methods utilize DNA ligation. DNA ligation is acommon molecular biology method for joining multiple DNA fragments.Ligases can seal single-strand nicks in duplex DNA, join two pieces withcomplementary “sticky” ends, and in some cases join two pieces withblunt, non-sticky ends. These methods find use in molecular cloning,nucleic acid diagnostics/detection, nucleic acid amplification, andrelated activities. Ligases and ligation methods are described, forexample, in U.S. Patent Application Publication No. 20120214208; U.S.Pat. No. 7,927,853; de Lumley et al., J. Mol. Biol. (2004) 339,1059-1075; and Rolland et al., FEMS Microbiol. Lett. (2004) 236,267-273.

Ligation of two strands of “sticky-ended” DNA is possible, where thesubstrates to be joined are already pre-positioned in the case ofsingle-strand nick sealing, and the affinity of sticky ends for eachother helps drive sticky-end ligation. However, since DNA ligationdepends upon the juxtaposition of the two substrates to be joined, DNAligation is more difficult in situations where the juxtaposition of thetwo substrates is more difficult, or the strands are likely to becomemis-aligned or to separate readily. For this reason, blunt-end and hightemperature ligations are two types of ligation that are particularlydifficult. Blunt-end ligation, however, depends upon random interactionsof the substrates. Two adjustments in blunt-end ligation are commonlymade to drive the substrate interactions: low temperature to slow downmolecular motion so that random interactions last longer and thus givethe ligase a better chance to join the fragments; and molecular crowdingreagents such as polyethylene glycol to increase the localconcentrations of substrates. Likewise, high temperature ligations arechallenging because the interactions of DNA substrates are briefer. Theleast efficient case is thus a high temperature blunt-end ligation inwhich molecular crowding reagents cannot be used.

A thermostable ligase, T4 DNA ligase, has been described that canperform blunt-end ligations; however, it is inactivated at temperatureabove approximately 45° C., so that the range of temperature at which T4DNA ligase is stable is relatively small. A few other examples ofligases that can be induced to join blunt-ended fragments are known, butthese ligases appear to do so only in the presence of highconcentrations of molecular crowding agents such as 50% polyethyleneglycol (PEG). However, the utility of ligases that require suchmolecular crowding agents is unclear, since 50% PEG which DNApolymerization (which is required for DNA amplification).

Accordingly, in order to facilitate the generation of amplified nucleicacids for the many and growing number of applications which useamplified nucleic acids, new methods and reagents for the amplificationof nucleic acids are desired.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

SUMMARY

Fusion proteins having thermostable blunt-end ligase activity areprovided. Such thermostable blunt-end ligases are useful for DNAamplification, sequencing, production of recombinant DNA (e.g., as aresult of such blunt-end fusions) and recombinant fusion proteins (e.g.,proteins encoded by recombinant DNA produced as a result of suchblunt-end fusions), and for other purposes. Many molecular biologyschemes involve ligases; such schemes would benefit from the ability toperform such schemes at elevated temperatures. Thermostable blunt-endDNA ligases disclosed herein are suitable for use in ligation schemeswhich would benefit from at least one elevated temperature incubation,such as an incubation at about 60-65° C. or higher, including incubationschemes at temperatures as high as about 94° C. A thermostable blunt-endligase as disclosed herein would be useful for such schemes.

In addition, thermostable blunt-end ligases as disclosed herein mayenable high temperature blunt-end ligation without the need formolecular crowding agents. Accordingly, thermostable blunt-end ligase asdisclosed herein may be useful for many nucleic acidligation-amplification schemes, including nucleic acidligation-amplification schemes which operate at a uniform temperature(e.g., at about 60° C. or higher), and including nucleic acidligation-amplification schemes which require temperature cycling such ascycling from, e.g., about 94° C. to about 60° C. (or higher) for one,two, three, or more cycles. Nucleic acid ligation-amplification schemeswhich may benefit from the use of thermostable blunt-end DNA ligasesdisclosed herein may operate at other temperatures as well, e.g.,depending on the temperature resistance characteristics of the novelfusion proteins disclosed herein and the requirements of the differencenucleic acid ligation-amplification schemes. Thermostable blunt-end DNAligases disclosed herein provide the ability to use high temperatures innucleic acid ligation-amplification schemes and thereby enable higherspecificity target amplification, for example, by permitting temperaturedenaturation of double-stranded DNA templates as well as specific primerbinding.

Accordingly, Applicant discloses a thermostable blunt-end nucleic acidligase comprising a fusion protein comprising a nucleic acid ligase anda nucleic acid binding protein, wherein said thermostable blunt-endnucleic acid ligase is suitable for use in a blunt-ended nucleic acidligation reaction performed at an elevated temperature. In embodiments,an elevated temperature comprises a temperature of about 60° C. orhigher. In embodiments, an elevated temperature comprises a temperatureof about 65° C., or about 70° C., or about 75° C., or about 80° C., orabout 85° C., or about 90° C., or about 95° C., or higher. Applicantfurther discloses an article of manufacture, comprising such athermostable blunt-end nucleic acid ligase, and a container. Inembodiments, the article of manufacture further comprises a buffer.Applicant further discloses a method of ligating blunt-end nucleic acidsat an elevated temperature, comprising using a thermostable blunt-endnucleic acid ligase as disclosed herein, wherein the use may be at atemperature of about 60° C. or higher. Applicant further discloses adevice for analyzing a sample containing nucleic acids, comprising athermostable blunt-end nucleic acid ligase comprising a fusion proteincomprising a nucleic acid ligase and a nucleic acid binding protein,wherein said thermostable blunt-end nucleic acid ligase is suitable foruse in a blunt-ended nucleic acid ligation reaction performed at about60° C. or higher.

In embodiments, Applicant discloses herein a thermostable blunt-end DNAligase comprising a fusion protein comprising a DNA ligase and a DNAbinding protein, wherein said thermostable blunt-end DNA ligase issuitable for use in a blunt-ended DNA ligation reaction performed at anelevated temperature, for example, at a temperature of about 60° C. orhigher. In embodiments, the thermostable blunt-end DNA ligase comprisesa T4 DNA ligase with an N-terminal p50 fusion.

In a particular embodiment, the thermostable blunt-end DNA ligasecomprises the amino acid sequence (SEQ ID NO: 1):

mghhhhhhhhhhssghiegrasadgpylqileqpkqrgfrfryvcegpshgglpgasseknkksypqvkicnyvgpakvivqlvtngknihlhahslvgkhcedgictvtagpkdmvvgfanlgilhvtkkkvfetlearmteacirgynpgllvhpdlaylqaegggdrqlgdrekelirqaalqqtkemdlsvvrimftaflpdstgsftrflepvvsdaiydskapnasnlkivrmdrtagcvtggeeiyllcdkvqkddiqirfyeeeenggywegfgdfsptdvhrqfaivfktpkykdinitkpasvfvqlrrksdletsepkpflyypeikdkeevqrkrqkgssgtsgggsgggmtleearkrvnelrdliryhnyryyvladpeisdaeydrllrelkeleerfpelkspdsptlqvgarpleatfrpvrhptrmysldnafnldelkafeerieralgrkgpfaytvehkvdglsvnlyyeegylvygatrgdgevgeevtqnlltiptiprrlkgvperlevrgevympieaflrineeleergerifknprnaaagslrqkdpritakrglratfyalglgleeveregvatqfallhwlkekgfpvehgyaravgaegveavyqdwlkkrralpfeadgvvvkldelalwrelgytaraprfaiaykfpaeeketrildvvfqvgrtgrvtpvgilepvflegsevsrvtlhnesyieeldirigdwvlvhkaggvipevlrvlkerrtgeerpirwpetcpecghrllkegkvhrcpnplcpakrfeairhfasrkamdiqglgekliernekglvkdvadlyrirkedlvglermgeksaqnllrqieeskkrglerilyalglpgygeylarnlaarfgnmdrileasleelleveevgeltaraileadpafrdlvalkeagvemeakekggealkgltfvitgelsrpreevkaltrrlgakvtdsysrktsylvvgenpgsklekaralgvptiteeelyrileartgkk aeelv.

A thermostable blunt-end DNA ligase as disclosed herein may comprise afusion protein which comprises a component selected from a peptidelinker, an N-terminal addition, a C-terminal addition, a tag peptide, aD-amino acid, and a peptide mimetic. In embodiments, a thermostableblunt-end DNA ligase as disclosed herein comprises a fusion proteinwhich comprises a component selected from a sugar and a polymer.

In embodiments, a thermostable blunt-end DNA ligase as disclosed hereinis suitable for use in a blunt-ended DNA ligation reaction performed atabout 75° C.

In embodiments, a thermostable blunt-end DNA ligase as disclosed hereinis capable of making concatamers upon multiple ligation events in ablunt-ended DNA ligation reaction performed at about 60° C. or higher.

In embodiments, the thermostable blunt-end DNA ligase as disclosedherein is suitable for use in a nucleic acid amplification scheme whichoperates at a uniform temperature of about 60° C. or higher.

In embodiments, the thermostable blunt-end DNA ligase as disclosedherein is suitable for use in a nucleic acid amplification scheme whichcomprises temperature cycling. In embodiments, such temperature cyclingcomprises temperature cycling to temperatures of about 60° C. or higher.In embodiments, such temperature cycling comprises temperature cyclingfrom about 94° C. to about 60° C. In embodiments, such temperaturecycling comprises two or more cycles of temperature cycling, and, inembodiments, comprises three or more cycles of temperature cycling.

Applicant further discloses a method of ligating blunt-end DNA atelevated temperature, comprising using a thermostable blunt-end DNAligase as disclosed herein, wherein the use may be at an elevatedtemperature. In embodiments, an elevated temperature comprises atemperature of about 60° C. or higher. In embodiments, an elevatedtemperature comprises a temperature of about 75° C.; and, in furtherembodiments, may comprise a temperature higher than about 75° C.

Applicant discloses herein an article of manufacture, comprising athermostable blunt-end DNA ligase and a container, wherein thethermostable blunt-end DNA ligase is a thermostable blunt-end DNA ligaseas disclosed herein. In embodiments, the article of manufacture furthercomprises a buffer.

Applicant discloses herein a device for analyzing a sample containingDNA, comprising a thermostable blunt-end DNA ligase, wherein thethermostable blunt-end DNA ligase is a thermostable blunt-end DNA ligaseas disclosed herein.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 shows an example of the results of a blunt-ended ligationreaction performed at 75° C. using a thermostable, blunt-end DNA ligaseas provided herein. The DNA substrate was a 49-bp duplex DNA made byannealing oligonucleotides EE0139 (SEQ ID NO:20) and EE0140 (SEQ ID NO:21).

DETAILED DESCRIPTION

Thermostable, blunt-end ligases are provided herein. As disclosedherein, a thermostable, blunt-end ligase may be prepared by the fusionof a DNA-binding protein to a thermostable ligase. Such ligases producedby these fusions have features and capabilities not provided by theirparent compounds alone; the different portions of these fusion proteinsprovide activities which, when combined, provide new capabilities andunexpectedly improved activity. The DNA-binding protein portion of suchfusion proteins is effective to increase the affinity of the ligase toDNA substrates, resulting in enhanced ligation in the challengingconditions of high temperature, blunt-end ligation. The DNA ligaseportion surprisingly retains its ability to ligate DNA when combinedwith a foreign protein (the DNA-binding protein portion). Together, thecombined portions unite in novel fusion proteins that are able to ligateblunt-ended DNA substrates even at high temperatures, providingincreased ligation activity at high temperatures unavailable by the useof the original, unmodified ligases.

Methods, reagents, devices, systems, and articles of manufacture usefulfor the practice of the methods, and for the use of reagents, devices,systems, and articles of manufacture disclosed herein, are described,for example, in U.S. Patent Application Ser. No. 61/800,606 (parent ofU.S. patent application Ser. No. 14/214,850, filed on Mar. 15, 2014),and U.S. Patent Application Ser. No. 61/800,925, the entire disclosuresof which are hereby incorporated by reference in their entireties.

DEFINITIONS

Before the present novel ligases and ligation methods are disclosed anddescribed, it is to be understood that the terminology used herein isfor the purpose of describing particular embodiments only and is notintended to be limiting. It is also to be understood that the presentdisclosure provides explanatory and exemplary descriptions and examples,so that, unless otherwise indicated, the molecules, compositions,assays, methods, and kits disclosed herein are not limited to thespecific embodiments described herein.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a salt” refers to a single salt or mixtures of differentsalts, and the like.

As used in the description herein and throughout the claims that follow,the meaning of “in” includes “in” and “on” unless the context clearlydictates otherwise.

As used in the description herein and throughout the claims that follow,the meaning of “or” includes both the conjunctive and disjunctive unlessthe context expressly dictates otherwise. Thus, the term “or” includes“and/or” unless the context expressly dictates otherwise.

In this specification and in the claims that follow, reference will bemade to a number of terms, which shall be defined to have the followingmeanings:

The term “moiety” as used herein refers to any particular composition ofmatter, e.g., a molecular fragment, an intact molecule, or a mixture ofmaterials.

The term “nucleic acid” refers to nucleotides and nucleosides which makeup, for example, deoxyribonucleic acid (DNA) macromolecules andribonucleic acid (RNA) macromolecules. Nucleic acids may be identifiedby the base attached to the sugar (e.g., deoxyribose or ribose); as usedherein, the following abbreviations for these bases are used torepresent nucleic acids in sequence listings identifying and describingtheir structures (either upper-case or lower-case may be used).

TABLE 1A Base (in Nucleic Acid) Letter Code Adenine A Thymine T GuanineG Cytosine C Uracil U

As used herein, a “polynucleotide” refers to a polymeric chaincontaining two or more nucleotides. “Polynucleotides” includes primers,oligonucleotides, nucleic acid strands, etc. A polynucleotide maycontain standard or non-standard nucleotides. Typically, apolynucleotide contains a 5′ phosphate at one terminus (“5′ terminus”)and a 3′ hydroxyl group at the other terminus (“3′ terminus) of thechain. The most 5′ nucleotide of a polynucleotide may be referred toherein as the “5′ terminal nucleotide” of the polynucleotide. The most3′ nucleotide of a polynucleotide may be referred to herein as the “3′terminal nucleotide” of the polynucleotide.

The term “downstream” as used herein in the context of a polynucleotidecontaining a 5′ terminal nucleotide and a 3′ terminal nucleotide refersto a position in the polynucleotide which is closer to the 3′ terminalnucleotide than a reference position in the polynucleotide. For example,in a primer having the sequence: 5′ ATAAGC 3′, the “G” is downstreamfrom the “T” and all of the “A” s.

The term “upstream” as used herein in the context of a polynucleotidecontaining a 5′ terminal nucleotide and a 3′ terminal nucleotide, refersto a position in the polynucleotide which is closer to the 5′ terminalnucleotide than a reference position in the polynucleotide. For example,in a primer having the sequence: 5′ ATAAGC 3′, the “T” is upstream fromthe “G”, the “C”, and the two “A” s closest to the “G”.

As used herein, “nucleic acid” includes both DNA and RNA, including DNAand RNA containing non-standard nucleotides. A “nucleic acid” containsat least one polynucleotide (a “nucleic acid strand”). A “nucleic acid”may be single-stranded or double-stranded.

As used herein, a nucleic acid molecule which is described as containingthe “sequence” of a template or other nucleic acid may also beconsidered to contain the template or other nucleic acid itself (e.g. amolecule which is described as containing the sequence of a template mayalso be described as containing the template), unless the contextclearly dictates otherwise.

As used herein, a “target” nucleic acid or molecule refers to a nucleicacid of interest. A target nucleic acid/molecule may be of any type,including single-stranded or double stranded DNA or RNA (e.g. mRNA).

As used herein, “complementary” sequences refer to two nucleotidesequences which, when aligned anti-parallel to each other, containmultiple individual nucleotide bases which pair with each other. It isnot necessary for every nucleotide base in two sequences to pair witheach other for sequences to be considered “complementary”. Sequences maybe considered complementary, for example, if at least 30%, 40%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of thenucleotide bases in two sequences pair with each other. In addition,sequences may still be considered “complementary” when the total lengthsof the two sequences are significantly different from each other. Forexample, a primer of 15 nucleotides may be considered “complementary” toa longer polynucleotide containing hundreds of nucleotides if multipleindividual nucleotide bases of the primer pair with nucleotide bases inthe longer polynucleotide when the primer is aligned anti-parallel to aparticular region of the longer polynucleotide.

As used herein, the term “isolated” as applied to proteins, nucleicacids, or other biomolecules refers to a molecule that has been purifiedor separated from a component of its naturally-occurring environment(e.g. a protein purified from a cell in which it was naturallyproduced). An “isolated” molecule may be in contact with other molecules(for example, as part of a reaction mixture). As used herein, “isolated”molecules also include recombinantly-produced proteins or nucleic acidswhich have an amino acid or nucleotide sequence which occurs naturally.“Isolated” nucleic acids include polypeptide-encoding nucleic acidmolecules contained in cells that ordinarily express the polypeptidewhere, for example, the nucleic acid molecule is at a chromosomallocation different from that of natural cells. In some embodiments,“isolated” polypeptides are purified to at least 50%, 60%, 70%, 80%,90%, 95%, 98%, or 100% homogeneity as evidenced by SDS-PAGE of thepolypeptides followed by Coomassie blue, silver, or other proteinstaining method.

The terms “polypeptide” and “protein” may be used interchangeably torefer to molecules comprised of amino acids linked by peptide bonds.Individual amino acids may be termed “residues” of a polypeptide orprotein. The amino acid sequences of polypeptides disclosed herein maybe identified by SEQ ID NO: presented as a string of letters, where theletters have the following meanings:

TABLE 1B Amino Acid 3-Letter Code 1-Letter Code Alanine Ala A ArginineArg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamic acidGlu E Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile ILeucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F ProlinePro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr YValine Val V

Naturally occurring amino acid residues are divided into groups based oncommon side-chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;(2) neutral hydrophilic: cys, ser, thr;(3) acidic: asp, glu;(4) basic: asn, gln, his, lys, arg;(5) residues that influence chain orientation: gly, pro; and(6) aromatic: trp, tyr, phe.

“Identical” or “identity,” as used herein in the context of two or morepolypeptide or polynucleotide sequences, can mean that the sequenceshave a specified percentage of residues that are the same over aspecified region. The percentage can be calculated by optimally aligningthe two sequences, comparing the two sequences over the specifiedregion, determining the number of positions at which the identicalresidue occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the specified region, and multiplying the result by 100to yield the percentage of sequence identity. In cases where the twosequences are of different lengths or the alignment produces one or morestaggered ends and the specified region of comparison includes only asingle sequence, the residues of the single sequence are included in thedenominator but not the numerator of the calculation.

“Homology” or “homologous” as used herein in the context of two or morepolypeptide or polynucleotide sequences, can mean that the sequenceshave a specified percentage of residues that are either i) the same, orii) conservative substitutions of the same residue, over a specifiedregion. Conservative substitutions include substitutions of one aminoacid by an amino acid of the same group, and include substitutions ofone amino acid by an amino acid identified in Table 1C as an exemplaryor as a preferred substitution. In determining homology of twosequences, identical residues and homologous residues are given equalweight. The percentage can be calculated by optimally aligning the twosequences, comparing the two sequences over the specified region,determining the number of positions at which either identical orhomologous residues occur in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the specified region, and multiplying the resultby 100 to yield the percentage of sequence homology. In cases where thetwo sequences are of different lengths or the alignment produces one ormore staggered ends and the specified region of comparison includes onlya single sequence, the residues of the single sequence are included inthe denominator but not the numerator of the calculation.

Variant or modified polypeptides may be prepared by substituting one ormore amino acid residues by a different one or more amino acid residuesin the amino acid sequence of the polypeptide. Conservativesubstitutions will entail exchanging one member of a group for anothermember of the same group; such changes tend not to significantly affectthe function of a polypeptide. Non-conservative substitutions willentail exchanging a member of one of these classes for another group.Substantial modifications of polypeptides may be accomplished withoutsignificantly affecting the functional characteristics of a polypeptideby selecting substitutions that maintain one or more of (a) thestructure of the polypeptide backbone in the area of the substitution,for example, as a sheet or helical conformation, (b) the charge orhydrophobicity of the molecule at the target site, or (c) the bulk ofthe side chain.

In particular embodiments, conservative substitutions of interest areshown in Table 1C under the headings of “exemplary substitutions” and“preferred substitutions.” Such conservative substitutions may notresult in significant changes in biological activity.

TABLE 1C Original Exemplary Preferred Residue SubstitutionsSubstitutions Ala (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn(N) gln; his; lys; arg gln Asp (D) glu glu Cys (C) ser ser Gln (Q) asnasn Glu (E) asp asp Gly (G) pro; ala ala His (H) asn; gln; lys; arg argIle (I) leu; val; met; ala; phe; norleucine leu Leu (L) norleucine; ile;val; met; ala; phe ile Lys (K) arg; gln; asn arg Met (M) leu; phe; ileleu Phe (F) leu; val; ile; ala; tyr leu Pro (P) ala ala Ser (S) thr thrThr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val(V) ile; leu; met; phe; ala; norleucine leu

Further substitutions may be performed, and suitable substitutions maybe identified by scanning amino acid analysis, in which a single aminoacid, or a very few amino acids, of an initial sequence are replaced.Among the preferred scanning amino acids are relatively small, neutralamino acids. Such amino acids include alanine, glycine, serine, andcysteine. Alanine is typically a preferred scanning amino acid amongthis group because it eliminates the side-chain beyond the beta-carbonand is less likely to alter the main chain conformation of the variant[Cunningham and Wells, Science, 244: 1081-1085 (1989)].

In addition, one or more amino acids in a sequence may be substituted bya non-conventional amino acid. Conservative replacements comprisingnon-conventional amino acids substitute the original amino acid with anon-conventional amino acid that resembles the original in one or moreof its, characteristic properties (e.g., charge, hydrophobicity, stearicbulk; for example, one may replace Val with Nval). The term“non-conventional amino acid” refers to amino acids other thanconventional amino acids, and includes, for example, isomers andmodifications of the conventional amino acids (e.g., D-amino acids),non-protein amino acids, post-translationally modified amino acids,enzymatically modified amino acids, constructs or structures designed tomimic amino acids (e.g., .alpha.,.alpha.-disubstituted amino acids,N-alkyl amino acids, lactic acid, .beta.-alanine, naphthylalanine,3-pyridylalanine, 4-hydroxyproline, 0-phosphoserine, N-acetylserine,N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, and norleucine),and peptides having the naturally occurring amide —CONH— linkagereplaced at one or more sites within the peptide backbone with anon-conventional linkage such as N-substituted amide, ester, thioamide,retropeptide (—NHCO—), retrothioamide (—NHCS—), sulfonamido (—SO.sub.2NH—), and/or peptoid (N-substituted glycine) linkages.

Homologous proteins and other protein variants may be providedsubstitution, insertion, deletion, or addition. Such substitution,insertion, deletion, or addition may include substitution, insertion,deletion, or addition of a single residue; may include substitution,insertion, deletion, or addition of two residues; and may includesubstitution, insertion, deletion, or addition of three, or of more,residues. For example, substitution may include replacing one, two,three, four, five, or more amino acid residues by a different residue.Substitution may be, or may include, conservative substitution. Forexample, insertion may include inserting one, two, three, four, five, ormore amino acid residues into an amino acid sequence. For example,deletion may include deleting one, two, three, four, five, or more aminoacid residues from an amino acid sequence. For example, addition mayinclude adding one, two, three, four, five, or more amino acid residuesat the N-terminal end, at the C-terminal end, or both, of an amino acidsequence.

A composition may include a buffer. Buffers include, without limitation,phosphate, citrate, ammonium, acetate, carbonate,tris(hydroxymethyl)aminomethane (TRIS), 3-(N-morpholino) propanesulfonicacid (MOPS), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO),2-(N-morpholino)ethanesulfonic acid (MES), N-(2-Acetamido)-iminodiaceticacid (ADA), piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES),N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES), cholamine chloride,N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES),2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid(TES), 4-(2-hydroxyethyl-1-piperazine ethanesulfonie acid (HEPES),acetamidoglycine, tricine(N-(2-Hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine), glycinamide, andbicine (2-(Bis(2-hydroxyethyl)amino)acetic acid) buffers. Buffersinclude other organic acid buffers in addition to the phosphate,citrate, ammonium, acetate, and carbonate buffers explicitly mentionedherein.

An article of manufacture may comprise a container; and a compositioncontained within the container, wherein the composition comprises athermostable ligase. An article of manufacture may comprise a container;and a composition contained within the container, wherein thecomposition comprises a blunt-end ligase. An article of manufacture maycomprise a container; and a composition contained within the container,wherein the composition comprises a thermostable blunt-end ligase. Thecontainers may be formed from a variety of materials such as glass orplastic, and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). The article of manufacture mayfurther comprise a label or package insert on or associated with thecontainer indicating that the composition may be used to ligateblunt-ended DNA.

Embodiments of methods and compositions provided herein may be describedwith reference to FIG. 1.

The novel thermostable blunt-end DNA ligases disclosed herein arecomprised of the fusion of a DNA-binding protein with a thermostableligase. Such fusion proteins as disclosed herein may further compriseother components, such as, for example, peptide linkers, N-terminal orC-terminal additions, tag peptides, and other amino acid sequences,including D-amino acids and peptide mimetics. In addition, fusionprotein as disclosed herein may further comprise other components, suchas, for example, sugars (e.g., the fusion proteins may be glycosylated),polymers such as polyethylene glycol (e.g., the fusion proteins may bePEGylated), organic moieties other than amino acids, and other additionslinked to the fusion proteins. The DNA-binding protein portion increasesthe affinity of the ligase to DNA substrates, resulting in enhancedligation in the challenging conditions of high temperature, blunt-endligation. Many combinations of binding proteins and ligases arepossible. For example, the binding protein may be N-terminal orC-terminal relative to the ligase.

It will be understood that multiple combinations of DNA ligases andDBA-binding proteins may be joined to produce the fusion proteinsdisclosed herein. For example, the following 16 combinations have beenconstructed (Table 2):

TABLE 2 Fusion Proteins Constructed

*Cells lacking a background color refer to the DNA-binding protein.Descriptions of CTF and p50 are given below. *Cells having a graybackground refers to the ligase. T4 = T4 phage, Tth = Thermusthermophilus, Pfu = Pyrococcus furiosus, Pfu C23 = Pfu with a C-terminal23-amino acid deletion.

Additional variations are possible, including: (1) different DNA-bindingproteins, including thermostable proteins; (2) other DNA ligases,including T4 DNA ligase variants engineered to be thermostable; (3)multiple DNA-binding proteins per ligase, or multiple ligases perDNA-binding protein; (4) transient, non-covalent linkages between theligase and DNA-binding protein, rather than a protein fusion, to betterenable multiple ligation events per ligase molecule; (5) dimers orhigher-order multimers of fusion proteins to increase the localconcentration of ligase once DNA substrates are bound; (6) differentdegrees of affinity between the DNA substrate and the DNA-bindingprotein—for example, using the natural target sequence of p50,GGGAATTCCC (SEQ ID NO: 6), in the DNA target, to enable low picomolaraffinity; (7) other nucleic acid modifying enzymes instead of DNA ligaseto perform other molecular reactions.

An exemplary method for producing the fusion proteins disclosed hereinmay be as follows. Fusion proteins may be made by inducing E. coli toexpress DNA constructs made by standard recombinant DNA methods,followed by standard chromatographic protein purification methods. Anaffinity tag such as polyhistidine may be employed to assist in theprotein purification. In the case of thermostable proteins, purificationmay be assisted by employing heat to denature most E. coli hostproteins. The purified fusion protein may be used in the same way that astandard, non-heat-stable ligase would be used in theapplication/reaction of choice, for example in a scheme that dependsupon ligation of DNA substrates to make a template for amplification.

The methods disclosed herein can be readily incorporated into and usedin device for processing a sample, or a system for processing a sample,which may be an automated assay device, or may be an automated assaysystem. Such assay devices and assay systems may comprise devices andsystems disclosed, for example, in U.S. Pat. No. 8,088,593; U.S. Pat.No. 8,380,541; U.S. patent application Ser. No. 13/769,798, filed Feb.18, 2013; U.S. patent application Ser. No. 13/769,779, filed Feb. 18,2013; U.S. patent application Ser. No. 13/244,947 filed Sep. 26, 2011;PCT/US2012/57155, filed Sep. 25, 2012; U.S. application Ser. No.13/244,946, filed Sep. 26, 2011; U.S. patent application Ser. No.13/244,949, filed Sep. 26, 2011; and U.S. Application Ser. No.61/673,245, filed Sep. 26, 2011, the disclosures of which patents andpatent applications are all hereby incorporated by reference in theirentireties.

Assay devices and assay systems, including devices for processing asample and systems for processing a sample, which may be automated assaydevices and automated assay systems, may be located and may be used at apoint of care location, and may be located and may be used at aplurality of point of care locations. Assay devices and assay systems,including devices for processing a sample and systems for processing asample, which may be automated assay devices and automated assaysystems, may be located and may be used at a point of care location, andmay be located and may be used at a plurality of point of servicelocations.

A sample processing device for performing assays and measurements asdisclosed herein may be located, and may perform such assays andmeasurements, at a point of care location, or a point of servicelocation, or other location. A sample processing system for performingassays and measurements as disclosed herein may be located, and mayperform such assays and measurements, at a point of care location, or apoint of service location, or other location.

A point of care system may be used at a point of service location, suchas a subject's location (e.g., home or business or sports event orsecurity screening or combat location), the location of a healthcareprovider (e.g., doctor), a pharmacy or retailer, a clinic, a hospital,an emergency room, a nursing home, a hospice care location, or alaboratory. A retailer may be a pharmacy (e.g., retail pharmacy,clinical pharmacy, hospital pharmacy), drugstore, chain store,supermarket, or grocer. Examples of retailers may include but are notlimited to Walgreen's, CVS Pharmacy, Duane Reade, Walmart, Target, RiteAid, Kroger, Costco, Kaiser Permanente, or Sears. In some situations, apoint of service system (including but not limited to point of caresystem) is deployed at any location that is designated for use by acertifying or licensing entity (e.g., a government certifying entity).In other situations, a point of service system may be used in orembedded in a transportation vehicle, such as a car, boat, truck, bus,airplane, motorcycle, van, traveling medical vehicle, mobile unit,ambulance, fire engine/truck, critical care vehicle, or other vehicleconfigured to transport a subject from one point to another. A samplecollection site may be at a sample acquisition site and/or healthassessment and/or treatment locations (which may include any of thesample collection sites described elsewhere herein including but notlimited to emergency rooms, doctors' offices, urgent care, tents forscreening (which may be in remote locations), a health care professionalwalking into someone's house to provide home care).

The system (device) or a combination of systems (devices) may belocated/positioned at strategic point of service locations. Locationsmay be selected and optimized based on a variety of objectives, such asbut not limited to disease prevalence, rates of disease development,projected disease rates, estimated risk of outbreaks, populationdemographics, government policies and regulations, customer, physicianand patient preferences, access to other technologies at said locations,safety and risk estimates, safety threats, etc. Devices can be relocatedon a periodic basis to improve overall utility on a frequent basis, suchas daily, weekly, monthly, annually, etc. Systems can be updated toimprove performance and/or add functionality. Systems can be updated ona module by module basis. System updates can occur via hardware and/orvia software. Systems can be updated with minimal downtime via featuresenabling blade and/or module extraction and insertion.

Additionally, a point of service location where a sample may becollected from a subject or provided by a subject may be a locationremote to an analyzing laboratory. The sample collection site may have aseparate facility from the laboratory. The sample may or may not becollected fresh from the subject at the point of service location.Alternatively, the sample may be collected from the subject elsewhereand brought to the point of service location. In some embodiments, nosample preparation step is provided on the sample before being providedto the device. For example, no slide needs to be prepped before a sampleis provided to the device. Alternatively, one or more sample preparationstep may be performed on the sample before being provided to the device.

A sample collection site at a point of service location may be a bloodcollection center, or any other bodily fluid collection center. Thesample collection site may be a biological sample collection center. Insome embodiments, a sample collection site may be a retailer. Otherexamples of sample collection sites may include hospitals, clinics,health care professionals' offices, schools, day-care centers, healthcenters, assisted living residences, government offices, travelingmedical care units, or the home. For example, a sample collection sitemay be a subject's home. A sample collection site may be any locationwhere a sample from the subject is received by the device. A collectionsite may be a moving location, such as on or with a patient or in amobile unit or vehicle or with a travelling doctor. Any location may bedesignated as a sample collection site. The designation may be made byany party, including but not limited to the laboratory, entityassociated with the laboratory, governmental agency, or regulatory body.Any description herein relating to sample collection site or point ofservice location may relate to or be applied to retailers, hospitals,clinics, or any other examples provided herein and vice versa.

A device may be part of a system, a component of which may be a sampleprocessing device. A device may be a sample processing device. A sampleprocessing device may be configured to facilitate collection of asample, prepare a sample for a clinical test, or effect a chemicalreaction with one or more reagents or other chemical or physicalprocessing, as disclosed herein. A sample processing device may beconfigured to obtain data from a sample. A sample processing device maybe configured to transmit data obtained from a sample. A sampleprocessing device may be configured to analyze data from a sample. Asample processing device may be configured to communicate with anotherdevice, or a laboratory, or an individual affiliated with a laboratory,to analyze data obtained from a sample.

Point of service systems described herein are configured to processsamples at a location where the point of service system is accessible bya user. In an example, a point of service system is located at asubject's home and a sample is collected from a subject and processed inthe subject's home. In another example, a point of service system islocated at a drug store and a sample is collected from a subject andprocessed in the drug store. In another example, a point of servicesystem is located at the location of a healthcare provider (e.g.,doctor's office) and a sample is collected from a subject and processedat the location of the healthcare provider. In another example, a pointof service system is located onboard a transportation system (e.g.,vehicle) and a sample is collected from a subject and processed on thetransportation system.

In some embodiments, post-sample processing analysis, includingdiagnosis and/or treatment, is performed by the point of service systemat the location of the point of service system. In other embodiments,post-sample processing analysis is performed remotely from a location inwhich a sample is collected and processed. In an example, post-sampleprocessing analysis is performed at the location of a healthcareprovider. In another example, post-sample processing analysis isperformed at the location of a processing system. In another example,post-sample processing analysis is performed on a server (e.g., on thecloud).

The post-sampling analysis may occur at a laboratory or by an entityaffiliated with a laboratory. A laboratory can be an entity or facilitycapable of performing a clinical test or analyzing collected data. Alaboratory can provide controlled conditions in which scientificresearch, experiments, and measurement can be performed. The laboratorycan be a medical laboratory or clinical laboratory where tests can bedone on clinical specimens, or analysis can occur on data collected fromclinical specimens, in order to get information about the health of apatient as pertaining to the diagnosis, prognosis, treatment, and/orprevention of disease. A clinical specimen may be a sample collectedfrom a subject. Preferably, a clinical specimen may be collected fromthe subject at a sample collection site that is at a separate facilityfrom the laboratory, as described in further detail elsewhere herein.The clinical specimen may be collected from the subject using a device,which is placed at a designated sample collection site or in or on thesubject.

A sample processing device may be configured to be placed in or on asubject. A sample processing device may be configured to accept a samplefrom a subject, either directly or indirectly. A sample may be, forexample, a blood sample (e.g., a sample obtained from a fingerstick, orfrom venipuncture, or an arterial blood sample), a urine sample, abiopsy sample, a tissue slice, stool sample, or other biological sample;a water sample, a soil sample, a food sample, an air sample; or othersample. A blood sample may comprise, e.g., whole blood, plasma, orserum. A sample processing device may receive a sample from the subjectthrough a housing of the device. The sample collection may occur at asample collection site, or elsewhere. The sample may be provided to thedevice at a sample collection site.

In some embodiments, a sample processing device may be configured toaccept or hold a cartridge. In some embodiments, a sample processingdevice may comprise a cartridge. The cartridge may be removable from thesample processing device. In some embodiments, a sample may be providedto the cartridge of the sample processing device. Alternatively, asample may be provided to another portion of a sample processing device.The cartridge and/or device may comprise a sample collection unit thatmay be configured to accept a sample.

A cartridge may include a sample, and may include reagents for use inprocessing or testing a sample, disposables for use in processing ortesting a sample, or other materials. Following placement of a cartridgeon, or insertion of a cartridge into, a sample processing device, one ormore components of the cartridge may be brought into fluid communicationwith other components of the sample processing device. For example, if asample is collected at a cartridge, the sample may be transferred toother portions of the sample processing device. Similarly, if one ormore reagents are provided on a cartridge, the reagents may betransferred to other portions of the sample processing device, or othercomponents of the sample processing device may be brought to thereagents. In some embodiments, the reagents or components of a cartridgemay remain on-board the cartridge. In some embodiments, no fluidics areincluded that require tubing or that require maintenance (e.g., manualor automated maintenance).

A sample or reagent may be transferred to a device, such as a sampleprocessing device. A sample or reagent may be transferred within adevice. Such transfer of sample or reagent may be accomplished withoutproviding a continuous fluid pathway from cartridge to device. Suchtransfer of sample or reagent may be accomplished without providing acontinuous fluid pathway within a device. In embodiments, such transferof sample or reagent may be accomplished by a sample handling system(e.g., a pipette); for example, a sample, reagent, or aliquot thereofmay be aspirated into an open-tipped transfer component, such as apipette tip, which may be operably connected to a movable unit whichtransfers the tip, with the sample, reagent, or aliquot thereofcontained within the tip, to a location on or within the sampleprocessing device. In some embodiments, a tip from the sample handlingsystem may be inserted at least partially into the sample vessel and/orcavity. The tip may be insertable and removable from the sample vesseland/or cavity. The sample, reagent, or aliquot thereof can be depositedat a location on or within the sample processing device. Sample andreagent, or multiple reagents, may be mixed using a sample handlingsystem in a similar manner. One or more components of the cartridge maybe transferred in an automated fashion to other portions of the sampleprocessing device, and vice versa.

A device, such as a sample processing device, may have a fluid handlingsystem (which may be part of a sample handling system). A fluid handlingsystem may perform, or may aid in performing, transport, dilution,extraction, aliquotting, mixing, and other actions with a fluid, such asa sample. In some embodiments, a fluid handling system may be containedwithin a device housing. A fluid handling system may permit thecollection, delivery, processing and/or transport of a fluid,dissolution of dry reagents, mixing of liquid and/or dry reagents with aliquid, as well as collection, delivery, processing and/or transport ofnon-fluidic components, samples, or materials. The fluid may be asample, a reagent, diluent, wash, dye, or any other fluid that may beused by the device, and may include, but not limited to, homogenousfluids, different liquids, emulsions, suspensions, and other fluids. Afluid handling system, including without limitation a pipette, may alsobe used to transport vessels (with or without fluid contained therein)around the device. The fluid handling system may dispense or aspirate afluid. The sample may include one or more particulate or solid matterfloating within a fluid.

In embodiments, a fluid handling system may comprise a pipette, pipettetip, syringe, capillary, or other component. The fluid handling systemmay have portion with an interior surface and an exterior surface and anopen end. The fluid handling system may comprise a pipette, which mayinclude a pipette body and a pipette nozzle, and may comprise a pipettetip. A pipette tip may or may not be removable from a pipette nozzle. Inembodiments, a fluid handling system may use a pipette mated with apipette tip; a pipette tip may be disposable. A tip may form afluid-tight seal when mated with a pipette. A pipette tip may be usedonce, twice, or more times. In embodiments, a fluid handling system mayuse a pipette or similar device, with or without a pipette tip, toaspirate, dispense, mix, transport, or otherwise handle the fluid. Thefluid may be dispensed from the fluid handling system when desired. Thefluid may be contained within a pipette tip prior to being dispensed,e.g., from an orifice in the pipette tip. In embodiments, or instancesduring use, all of the fluid may be dispensed; in other embodiments, orinstances during use, a portion of the fluid within a tip may bedispensed. A pipette may selectively aspirate a fluid. The pipette mayaspirate a selected amount of fluid. The pipette may be capable ofactuating stirring mechanisms to mix the fluid within the tip or withina vessel. The pipette may incorporate tips or vessels creatingcontinuous flow loops for mixing, including of materials or reagentsthat are in non-liquid form. A pipette tip may also facilitate mixtureby metered delivery of multiple fluids simultaneously or in sequence,such as in 2-part substrate reactions.

The fluid handling system may include one or more fluidically isolatedor hydraulically independent units. For example, the fluid handlingsystem may include one, two, or more pipette tips. The pipette tips maybe configured to accept and confine a fluid. The tips may be fluidicallyisolated from or hydraulically independent of one another. The fluidcontained within each tip may be fluidically isolated or hydraulicallyindependent from one fluids in other tips and from other fluids withinthe device. The fluidically isolated or hydraulically independent unitsmay be movable relative to other portions of the device and/or oneanother. The fluidically isolated or hydraulically independent units maybe individually movable. A fluid handling system may comprise one ormore base or support. A base or support may support one or more pipetteor pipette units. A base or support may connect one or more pipettes ofthe fluid handling system to one another.

A sample processing device may be configured to perform processing stepsor actions on a sample obtained from a subject. Sample processing mayinclude sample preparation, including, e.g., sample dilution, divisionof a sample into aliquots, extraction, contact with a reagent,filtration, separation, centrifugation, or other preparatory orprocessing action or step. A sample processing device may be configuredto perform one or more sample preparation action or step on the sample.Optionally, a sample may be prepared for a chemical reaction and/orphysical processing step. A sample preparation action or step mayinclude one or more of the following: centrifugation, separation,filtration, dilution, enriching, purification, precipitation,incubation, pipetting, transport, chromatography, cell lysis, cytometry,pulverization, grinding, activation, ultrasonication, micro columnprocessing, processing with magnetic beads, processing withnanoparticles, or other sample preparation action or steps. For example,sample preparation may include one or more step to separate blood intoserum and/or particulate fractions, or to separate any other sample intovarious components. Sample preparation may include one or more step todilute and/or concentrate a sample, such as a blood sample, or otherbiological samples. Sample preparation may include adding ananti-coagulant or other ingredients to a sample. Sample preparation mayalso include purification of a sample. In embodiments, all sampleprocessing, preparation, or assay actions or steps are performed by asingle device. In embodiments, all sample processing, preparation, orassay actions or steps are performed within a housing of a singledevice. In embodiments, most sample processing, preparation, or assayactions or steps are performed by a single device, and may be performedwithin a housing of a single device. In embodiments, many sampleprocessing, preparation, or assay actions or steps are performed by asingle device, and may be performed within a housing of a single device.In embodiments, sample processing, preparation, or assay actions orsteps may be performed by more than one device.

A sample processing device may be configured to run one or more assay ona sample, and to obtain data from the sample. An assay may include oneor more physical or chemical treatments, and may include running one ormore chemical or physical reactions. A sample processing device may beconfigured to perform one, two or more assays on a small sample ofbodily fluid. One or more chemical reaction may take place on a samplehaving a volume, as described elsewhere herein. For example one or morechemical reaction may take place in a pill having less than femtolitervolumes. In an instance, the sample collection unit is configured toreceive a volume of the bodily fluid sample equivalent to a single dropor less of blood or interstitial fluid. In embodiments, the volume of asample may be a small volume, where a small volume may be a volume thatis less than about 1000 μL, or less than about 500 μL, or less thanabout 250 μL, or less than about 150 μL, or less than about 100 μL, orless than about 75 μL, or less than about 50 μL, or less than about 40μL, or less than about 20 μL, or less than about 10 μL, or other smallvolume. In embodiments, all sample assay actions or steps are performedon a single sample. In embodiments, all sample assay actions or stepsare performed by a single device. In embodiments, all sample assayactions or steps are performed within a housing of a single device. Inembodiments, most sample assay actions or steps are performed by asingle device, and may be performed within a housing of a single device.In embodiments, many sample assay actions or steps are performed by asingle device, and may be performed within a housing of a single device.In embodiments, sample processing, preparation, or assay actions orsteps may be performed by more than one device.

A sample processing device may be configured to perform a plurality ofassays on a sample. In embodiments, a sample processing device may beconfigured to perform a plurality of assays on a single sample. Inembodiments, a sample processing device may be configured to perform aplurality of assays on a single sample, where the sample is a smallsample. For example, a small sample may have a sample volume that is asmall volume of less than about 1000 μL, or less than about 500 μL, orless than about 250 μL, or less than about 150 μL, or less than about100 μL, or less than about 75 μL, or less than about 50 μL, or less thanabout 40 μL, or less than about 20 μL, or less than about 10 μL, orother small volume. A sample processing device may be capable ofperforming multiplexed assays on a single sample. A plurality of assaysmay be run simultaneously; may be run sequentially; or some assays maybe run simultaneously while others are run sequentially. One or morecontrol assays and/or calibrators (e.g., including a configuration witha control of a calibrator for the assay/tests) can also be incorporatedinto the device; control assays and assay on calibrators may beperformed simultaneously with assays performed on a sample, or may beperformed before or after assays performed on a sample, or anycombination thereof. In embodiments, all sample assay actions or stepsare performed by a single device. In embodiments, all of a plurality ofassay actions or steps are performed within a housing of a singledevice. In embodiments, most sample assay actions or steps, of aplurality of assays, are performed by a single device, and may beperformed within a housing of a single device. In embodiments, manysample assay actions or steps, of a plurality of assays, are performedby a single device, and may be performed within a housing of a singledevice. In embodiments, sample processing, preparation, or assay actionsor steps may be performed by more than one device.

In embodiments, all of a plurality of assays may be performed in a shorttime period. In embodiments, such a short time period comprises lessthan about three hours, or less than about two hours, or less than aboutone hour, or less than about 40 minutes, or less than about 30 minutes,or less than about 25 minutes, or less than about 20 minutes, or lessthan about 15 minutes, or less than about 10 minutes, or less than about5 minutes, or less than about 4 minutes, or less than about 3 minutes,or less than about 2 minutes, or less than about 1 minute, or othershort time period.

A device may be capable of performing all on-board steps (e.g., steps oractions performed by a single device) in a short amount of time. Adevice may be capable of performing all on-board steps on a singlesample in a short amount of time. For example, from sample collectionfrom a subject to transmitting data and/or to analysis may take about 3hours or less, 2 hours or less, 1 hour or less, 50 minutes or less, 45minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes orless, 15 minutes or less, 10 minutes or less, 5 minutes or less, 4minutes or less, 3 minutes or less, 2 minutes or less, or 1 minute orless. The amount of time from accepting a sample within the device totransmitting data and/or to analysis from the device regarding such asample may depend on the type or number of steps, tests, or assaysperformed on the sample. The amount of time from accepting a samplewithin the device to transmitting data and/or to analysis from thedevice regarding such a sample may take about 3 hours or less, 2 hoursor less, 1 hour or less, 50 minutes or less, 45 minutes or less, 40minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes orless, 10 minutes or less, 5 minutes or less, 4 minutes or less, 3minutes or less, 2 minutes or less, or 1 minute or less.

A device or system as disclosed herein, such as, e.g., a point ofservice device or a point of service system, may process a sampleaccording to a test order or schedule. In some situations, a feedbackloop (coupled with sensors) enables the point of service device orsystem to monitor the progress of sample processing and maintain oralter the test order or schedule. In an example, if the device or systemdetects that processing is taking longer than the predetermined amountof time set forth in the schedule, the device or system speeds upprocessing or adjusts any parallel processes, such as sample processingin another module of the device or system. The feedback loop permitsreal-time or pseudo-real time (e.g., cached) monitoring. In somesituations, the feedback loop may provide permit reflex testing, whichmay cause subsequent tests, assays, preparation steps, and/or otherprocesses to be initiated after starting or completing another testand/or assay or sensing one or more parameter. Such subsequent tests,assays, preparation steps, and/or other processes may be initiatedautomatically without any human intervention.

In some embodiments, the point of service system may stick to apre-determined test order or schedule based on initial parameters and/ordesired effects. In other embodiments, the schedule and/or test ordermay be modified on the fly. The schedule and/or test order may bemodified based on one or more detected conditions, one or moreadditional processes to run, one or more processes to no longer run, oneor more processes to modify, one or more resource/component utilizationmodifications, one or more detected error or alert condition, one ormore unavailability of a resource and/or component, one or moresubsequent input or sample provided by a user, external data, or anyother reason.

In some examples, one or more additional samples may be provided to adevice after one or more initial samples are provided to the device. Theadditional samples may be from the same subject or different subjects.The additional samples may be the same type of sample as the initialsample or different types of samples (e.g., blood, tissue). Theadditional samples may be provided prior to, concurrently with, and/orsubsequent to processing the one or more initial samples on the device.The same and/or different tests or desired criteria may be provided forthe additional samples, as opposed to one another and/or the initialsamples. The additional samples may be processed in sequence and/or inparallel with the initial samples. The additional samples may use one ormore of the same components as the initial samples, or may use differentcomponents. The additional samples may or may not be requested in viewof one or more detected condition of the initial samples.

In some embodiments, the system accepts a sample with the aid of asample collection module, such as a lancet, scalpel, or fluid collectionvessel. The system then loads or accesses a protocol for performing oneor more processing routines from a plurality of potential processingroutines. In an example, the system loads a centrifugation protocol andcytometry protocol. In some embodiments, the protocol may be loaded froman external device to a sample processing device. Alternatively, theprotocol may already be on the sample processing device. The protocolmay be generated based on one or more desired criteria and/or processingroutines. In one example, generating a protocol may include generating alist of one or more subtasks for each of the input processes. In someembodiments, each subtask is to be performed by a single component ofthe one or more devices. Generating a protocol may also includegenerating the order of the list, the timing and/or allocating one ormore resources.

In an embodiment, a protocol provides processing details orspecifications that are specific to a sample or a component in thesample. For instance, a centrifugation protocol may include rotationalvelocity and processing time that is suited to a predetermined sampledensity, which enables density-dependent separation of a sample fromother material that may be present with a desirable component of thesample.

A protocol is included in the system, such as in a protocol repositoryof the system, or retrieved from another system, such as a database, incommunication with the system. In an embodiment, the system is inone-way communication with a database server that provides protocols tothe system upon request from the system for one or more processingprotocols. In another embodiment, the system is in two-way communicationwith a database server, which enables the system to upload user-specificprocessing routines to the database server for future use by the user orother users that may have use for the user-specific processing routines.

In some cases, a processing protocol is adjustable by a user. In anembodiment, a user may generate a processing protocol with the aid of aprotocol engine that provides the user one or more options geared towardtailoring the protocol for a particular use. The tailoring may occurprior to use of the protocol. In some embodiments, the protocol may bemodified or updated while the protocol is in use.

With the aid of a protocol, a system processes a sample, which mayinclude preparing the sample, assaying the sample and detecting one ormore components of interest in the sample. In some cases, the systemperforms data analysis with respect to the sample or a plurality ofsample after processing. In other cases, the system performs dataanalysis during processing. In some embodiments, data analysis isperformed on-board—that is, on the system. In other embodiments, dataanalysis is performed using a data analysis system that is external tothe system. In such a case, data is directed to the analysis systemwhile the sample is being processed or following processing.

A device may be configured to prepare a sample for disposal, or todispose of a sample, such as a biological sample, following processingor assaying of a sample.

In embodiments, a sample processing device may be configured to transmitdata obtained from a sample. In embodiments, a sample processing devicemay be configured to communicate over a network. A sample processingdevice may include a communication module that may interface with thenetwork. A sample processing device may be connected to the network viaa wired connection or wirelessly. The network may be a local areanetwork (LAN) or a wide area network (WAN) such as the Internet. In someembodiments, the network may be a personal area network. The network mayinclude the cloud. The sample processing device may be connected to thenetwork without requiring an intermediary device, or an intermediarydevice may be required to connect a sample processing device to anetwork. A sample processing device may communicate over a network withanother device, which may be any type of networked device, including butnot limited to a personal computer, server computer, or laptop computer;personal digital assistants (PDAs) such as a Windows CE device; phonessuch as cellular phones, smartphones (e.g., iPhone, Android, Blackberry,etc.), or location-aware portable phones (such as GPS); a roamingdevice, such as a network-connected roaming device; a wireless devicesuch as a wireless email device or other device capable of communicatingwireless with a computer network; or any other type of network devicethat may communicate possibly over a network and handle electronictransactions. Such communication may include providing data to a cloudcomputing infrastructure or any other type of data storageinfrastructure which may be accessed by other devices.

A sample processing device may provide data regarding a sample to, e.g.,a health care professional, a health care professional location, such asa laboratory, or an affiliate thereof. One or more of a laboratory,health care professional, or subject may have a network device able toreceive or access data provided by the sample processing device. Asample processing device may be configured to provide data regarding asample to a database. A sample processing device may be configured toprovide data regarding a sample to an electronic medical records system,to a laboratory information system, to a laboratory automation system,or other system or software. A sample processing device may provide datain the form of a report.

A laboratory, device, or other entity or software may perform analysison data regarding a sample in real-time. A software system may performchemical analysis and/or pathological analysis, or these could bedistributed amongst combinations of lab, clinical, and specialty orexpert personnel. Analysis may include qualitative and/or quantitativeevaluation of a sample. Data analysis may include a subsequentqualitative and/or quantitative evaluation of a sample. Optionally, areport may be generated based on raw data, pre-processed data, oranalyzed data. Such a report may be prepared so as to maintainconfidentiality of the data obtained from the sample, the identity andother information regarding the subject from whom a sample was obtained,analysis of the data, and other confidential information. The reportand/or the data may be transmitted to a health care professional. Dataobtained by a sample processing device, or analysis of such data, orreports, may be provided to a database, an electronic medical recordssystem, to a laboratory information system, to a laboratory automationsystem, or other system or software.

EXAMPLES Example 1

Examples of individual protein components suitable for use in providingfusion proteins as disclosed herein include (the amino acid sequencesare provided using the one-letter code for amino acids):

TABLE 3 SEQ ID Protein Name Protein Sequence NO: p50 DNA-bindingadgpylgileqpkgrgfrfryvcegpshgglpgasseknkksy  2 protein (fragmentpqvkicnyvgpakvivqlvtngknihlhahslvgkhcedgict from the human NF-vtagpkdmvvgfanlgilhvtkkkvfetlearmteacirgynp kappa-B protein,gllvhpdlaylqaegggdrqlgdrekelirciaalqqtkemdls accession numbervvrlmftaflpdstgsftrrlepvvsdaiydskapnasnlkiv NP 003989, aminormdrtagcvtggeeiyllcdkvqkddigirfyeeeenggvweg acids 40-366)fgdfsptdvhrgfaivfktpkykdinitkpasvfvqlrrksdl etsepkpflyypeikdkeevqrkrqkTth DNA ligase mtleearkrvnelrdliryhnyryyvladpeisdaeydrllre  3 (Thermuslkeleerfpelkspdsptlqvgarpleatfrpvrhptrmysld Thermophilus strainnafnldelkafeerieralgrkgpfaytvehkvdglsvnlyye HB8, accessionegylvygatrgdgevgeevtqnlltiptiprrlkgyperlevr YP_144363.1)gevympieaflrineeleergerifknprnaaagslrqkdpritakrglratfyalglgleeveregvatqfallhwlkekgfpvehgyaravgaegveavygdwlkkrralpfeadgyvvkldelalwrelgytaraprfaiaykfpaeeketrlldvvfqvgrtgrvtpvgilepvflegsevsrvtlhnesyieeldirigdwvlvhkaggvipevlrvlkerrtgeerpirwpetcpecghrllkegkvhrcpnplcpakrfeairhfasrkamdigglgeklierllekglykdvadlyrlrkedlyglermgeksagnllrgieeskkrglerllyalglpgvgevlarnlaarfgnmdrlleasleelleveevgeltarailetlkdpafrdlvrrlkeagvemeakekggealkgltfvitgelsrpreevkallrrlgakvtdsysrktsylvvgenpgsklekaralgyptlteeelyrlleartgkkaeelv His10-containing mghhhhhhhhhhssghiegras 4 leader Flexible glycine- gssgtsgggsggg  5 rich sequenceCTF DNA-binding sptsymspslpaldwqlpshsgpyelrievqpkshhrahyete  7protein (a hybrid gsrgavkasagghpivqlhgylenepltlqlfigtaddrllrpfrom the murine hafyqvhritgktysttsheiilsntkvleipllpennmraiiNFATc1 protein, dcagilklrnsdielrkgetdigrkntrvrlyfrvhipqpngraccession number tlslciasnlkivrmdrtagcvtggeeiyllcdkvqkddigirfNP_058071, amino yeeeenggvwegfgdfsptdvhrgfaivfktpkykdinitkpaacids 390-506; svfvqlrrksdletsepkpflyypeikdkeevqrkrqk followed by analanine spacer; followed by a fragment from the human NF-kappa-Bprotein, accession number NP_003989, amino acids 249-366) T4 DNA ligasemilkilneiasigstkqkgaileknkdnellkrvyrltysrgl  8 (accession numberqyyikkwpkpgiatqsfgmltltdmldfieftlatrkltgnaa NP_049813)ieeltgyitdgkkddvevlrrvmmrdlecgasysiankvwpg1ipeqpqmlassydekginknikfpafaqlkadgarcfaevrgdelddvrllsragneylgldllkeelikmtaearqihpegvlidgelvyheqvkkepegldflfdaypenskakefaevaesrtasngiankslkgtisekeaqcmkfqvwdyvplveiyslpafrlkydvrfsklegmtsgydkvilienqvvnnldeakviykkyidgglegiilknidglwenarsknlykfkevidvdlkivgiyphrkdptkaggfilesecgkikvnagsglkdkagvksheldrtrimenqnyyigkilececngwlksdgrtdyvklflpiairlredktkant fedvfgdfhevtglPfu DNA ligase mrylelaglyqklekttmkliktrlvadflkkvpddhlefipy  9(Pyrococcus furiosus lilgdvfpewderelgvgekllikavamatgidaneiensvkdstrain ST04, tgdlgesialavkkrkqksffscipltikrvyqtivkvaettge accessiongsgekkmkylanlfmdaepieakyiartvlgtmrtgvaegllr YP_006355162)daialafhvkvelveraymltsdfgfvakvaklegneglakvqvgigkpikpmlaqqaanikeallemggeaefeikydgarvqvhkdgdkiivysrrlenvtraipeivealkqsvkpnkaivegelvaigedgrplpfqyvlrrfrrkhniqemmkkiplelnlfdvlyvdgesmidvkfidrrkkleeiiepngkikvaenlitkkveeaeafykkalemgheglmakrldatyepgnrgkkwlkikptmenldlviigaewgegrrahllgsfilgaydpetgeflevgkvgsgftdedlveftkmlkpliikeegkrvwiepkivievtycleigkspkyksgfalrfpryvalrddkgpedadtieriaglyelgermkgkv Pfu C23 DNA ligasemrylelaglyqklekttmkliktrlvadflkkvpddhlefipy 10 (C-terminal 23-aminolilgdvfpewderelgvgekllikavamatgidaneiensvkd acid deletion oftgdlgesialavkkrkqksffscipltikrvyqtivkvaettge Pfu)gsgekkmkylanlfmdaepieakyiartvlgtmrtgvaegllrdaialafhvkvelveraymltsdfgfvakvaklegneglakvqvgigkpikpmlaqqaanikeallemggeaefeikydgarvqvhkdgdkiivysrrlenvtraipeivealkqsvkpnkaivegelvaigedgrplpfqyvlrrfrrkhniqemmkkiplelnlfdvlyvdgesmidvkfidrrkkleeiiepngkikvaenlitkkveeaeafykkalemgheglmakrldatyepgnrgkkwlkikptmenldlviigaewgegrrahllgsfilgaydpetgeflevgkvgsgftdedlveftkmlkpliikeegkrvwiepkivievtycleigkspky ksgfalrfpryvalrddkgpHIS-rich leader, p50 Mghhhhhhhhhhssghiegrasadgpylgileqpkgrgfrfry 11DNA-binding protein, vcegpshgglpgasseknkksypqvkicnyvgpakvivqlvtnglycine-rich linker gknihlhahslvgkhcedgictvtagpkdmvvgfanlgilhvtkkkvfetlearmteacirgynpgllvhpdlaylqaegggdrqlgdrekelirciaalqqtkemdlsvvrlmftaflpdstgsftrrlepvvsdaiydskapnasnlkivrmdrtagcvtggeeiyllcdkvqkddigirfyeeeenggvwegfgdfsptdvhrgfaivfktpkykdinitkpasvfvqlrrksdletsepkpflyypeikdkeevq rkrqkgssgtsgggsgggHIS-rich leader, CTF Mghhhhhhhhhhssghiegrassptsymspslpaldwqlpshs 12DNA-binding protein, gpyelrievqpkshhrahyetegsrgavkasagghpivqlhgyglycine-rich linker lenepltlqlfigtaddrllrphafyqvhritgktvsttsheiilsntkvleipllpennmraiidcagilklrnsdielrkgetdigrkntrvrlvfrvhipqpngrtlslqasnlkivrmdrtagcvtggeeiyllcdkvqkddigirfyeeeenggvwegfgdfsptdvhrgfaivfktpkykdinitkpasvfvqlrrksdletsepkpflyypeikdkeevqrkrqkgssgtsgggsggg

The following provides an example of a fusion protein providingthermostable blunt-end DNA ligase activity:

-   -   p50-Tth ligase fusion, Theranos construct EE0217, e.g. protein        prep RDP0078, consists of a His10-containing leader        (mghhhhhhhhhhssghiegras, SEQ ID NO: 4), p.50 (shown below in        italic text, beginning with adgpy . . . and ending with . . .        rkrqk, SEQ ID NO: 2), a flexible glycine-rich sequence        (gssgtsgggsggg, SEQ ID NO: 5), and the Tth DNA ligase (shown        below in underlined text, SEQ ID NO: 3). This fusion protein has        the following sequence (SEQ ID NO: 1):

mghhhhhhhhhhssghiegrasadgpylqileqpkqrgfrfryvcegpshgglpgasseknkksypqvkicnyvgpakvivqlvtngknihlhahslygkhcedgictvtagpkdmvvgfanlgilhytkkkvfetlearmteacirgynpgllvhpdlaylqaegggdrqlgdrekelirqaalqqtkemdlsvvrlmftaflpdstgsftrrlepyysdaiydskapnasnlkivrmdrtagcvtggeeiyllcdkvqkddiqirfyeeeenggvwegfgdfsptdvhrqfaivfktpkykdinitkpasvfvqlrrksdletsepkpflyypeikdkeevqrkrqkgssgtsgggsgggmtleearkrynelrdliryhrwryyyladpeisdaeydrllrelkeleerfpelkspdsptlqvgarpleatfrpyrhptrmysldnafnldelkafeerieralgrkgpfaytvehkydglsvnlyyeegylvygatrgdgevgeevtqnlltiptiprrlkgyperlevrgevympieaflrlneeleergerifknprnaaagslrqkdpritakrglratfyalglgleeveregvatqfallhwlkekgfpvehgyaravgaegveavyqdwlkkrralpfeadgyvvkldelalwrelgytaraprfaiaykfpaeeketrlldvvfqvgrtgrytpvgilepvflegsevsrvtlhnesyieeldirigdwvlvhkaggvipevlrylkerrtgeerpirwpetcpecghrllkegkyhrcpnplcpakrfeairhfasrkamdiqglgeklierllekglykdvadlyrlrkedlyglermgeksaqnllrqieeskkrglerllyalglpgygevlarnlaarfgnmdrlleasleelleveevgeltarailetlkdpafrdlyrrlkeagvemeakekggealkgltfyitgelsrpreevkallrrlgakvtdsysrktsylvvgenpgsklekaralgyptlteeelyrllear tgkkaeelv

The fusion protein sequence SEQ ID NO: 1 presented above is an exampleof a fusion protein that may be represented as “4-2-5-3” in which “4”represents SEQ ID NO: 4; “2” represents SEQ ID NO: 2; “5” represents SEQID NO: 5; and “3” represents SEQ ID NO: 3. Such representationsindicate, in a N-terminal to C-terminal orientation, linear fusionproteins comprised of fusions of the amino acid sequence indicated bythe numbers (which themselves indicate the corresponding SEQ ID NO:s).Further such representations of fusion proteins are presented below.

An example of a blunt-ended ligation reaction performed at 75° C. isprovided below. The DNA substrate was a 49-bp duplex DNA made byannealing oligonucleotides EE0139 (SEQ ID NO: 20) and EE0140 (SEQ ID NO:21). This blunt-ended duplex was capable of making concatamers uponmultiple ligation events. The results of the reaction products wereseparated by size on an agarose gel, as shown in FIG. 1, demonstratethat: (1) Tth ligase alone (lanes 2-3), although it is known to becapable of sealing DNA nicks at 75° C., performed very little or noblunt ligation in these conditions; (2) T4 DNA ligase with an N-terminalp50 fusion (lanes 4-5) also performed very little blunt ligation inthese conditions, although there was some evidence of more ligation thanfor Tth alone. This observation was believed to be surprising given thetemperature sensitivity of T4 ligase alone; (3) Tth DNA ligase with anN-terminal p50 fusion (lanes 6-7) demonstrated a much higher level ofblunt-end ligation at 75° C. Thus, the results shown in FIG. 1demonstrate that fusion proteins as disclosed herein provide improvedblunt-end ligation activity at high temperature. Fusion proteinshomologous to SEQ ID NO: 1 are believed to provide improved blunt-endligation activity at high temperature. Thus, fusion proteins havinggreater than 90%, or greater than 95% or greater than 99%, sequencehomology to SEQ ID NO: 1 are believed to provide blunt-end DNA ligationactivity at high temperature.

Example 2

The fusion protein of SEQ ID NO: 1 (and homologous molecules) is anexample of a thermostable blunt-end DNA ligase. Examples of furtherfusion proteins having similar structural characteristics are listed inthe following table (Table 4). It is believed that these further fusionproteins share not only structural characteristics, but also functionalcharacteristics as well; thus, the fusion proteins listed in Table 4 arebelieved to be thermostable blunt-end ligases. In addition, proteinshomologous to the fusion proteins of Table 4 believed to be thermostableblunt-end ligases. The first-listed example, SEQ ID NO: 1, has beendiscussed above, and results of ligation experiments using SEQ ID NO: 1are shown in FIG. 1. As discussed above, SEQ ID NO: 1 is a combinationof a HIS 10-containing leader sequence (SEQ ID NO: 4), the DNA-bindingprotein p50 (SEQ ID NO: 2), a flexible glycine-rich sequence (SEQ ID NO:5) and Tth DNA ligase (SEQ ID NO: 3), in which the amino acid sequenceSEQ ID NO: 4 is covalently linked to the amino acid sequence SEQ ID NO:2 which is covalently linked to the amino acid sequence SEQ ID NO: 5which is covalently linked to the amino acid sequence SEQ ID NO: 3; thiscombination is termed “4-2-5-3” in the left-most column of Table 4. (Theterm “4-2-5-3” indicates a linear fusion protein in which sequences SEQID NO: 4, SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO: 3 are linked aswritten, from N-terminal (left) to C-terminal (right).) Furthercombinations of such components illustrate some of the many possiblefusion proteins having features as disclosed herein. The other fusionproteins listed in Table 4 include further combinations of leadersequences (SEQ ID NO: 4) with a DNA-binding protein (e.g., SEQ ID NO: 2or SEQ ID NO: 7), a flexible glycine-rich sequence (SEQ ID NO: 5), and aDNA ligase (SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10).Thus, exemplary fusion proteins disclosed in Table 4 include an aminoacid sequence of a histidine-rich leader sequence, a DNA ligase, and aflexible glycine-rich linker sequence (i.e., SEQ ID NO: 1 lor SEQ ID NO:12) covalently linked to a ligase (e.g., SEQ ID NO: 3, SEQ ID NO: 8, SEQID NO: 9, or SEQ ID NO: 10). These exemplary fusion proteins arebelieved to provide yet further thermostable blunt-ended nucleic acidligases.

TABLE 4 Fusion Protein Formed by  SEQ ID SEQ ID NOs: Protein SequenceNO: 4-2-5-3 mghhhhhhhhhhssghiegrasadgpylgileqpkgrgfrfryvcegps  1hgglpgasseknkksypqvkicnyvgpakvivqlvtngknihlhahslvgkhcedgictvtagpkdmvvgfanlgilhvtkkkvfetlearmteacirgynpgllvhpdlaylqaegggdrqlgdrekelirciaalqqtkemdlsvvrlmftaflpdstgsftrrlepvvsdaiydskapnasnlkivrmdrtagcvtggeeiyllcdkvqkddigirfyeeeenggvwegfgdfsptdvhrcifaivfktpkykdinitkpasvfvqlrrksdletsepkpflyypeikdkeevqrkrqkgssgtsgggsgggmtleearkrvnelrdliryhnyryyvladpeisdaeydrllrelkeleerfpelkspdsptlqvgarpleatfrpvrhptrmysldnafnldelkafeerieralgrkgpfaytvehkvdglsvnlyyeegvlvygatrgdgevgeevtqnlltiptiprrlkgvperlevrgevympieaflrineeleergerifknprnaaagslrqkdpritakrglratfyalglgleeveregvatqfallhwlkekgfpvehgyaravgaegveavyqdwlkkrralpfeadgvvvkldelalwrelgytaraprfaiaykfpaeeketrlldvvfqvgrtgrvtpvgilepvflegsevsrvtlhnesyieeldirigdwvlvhkaggvipevlrvlkerrtgeerpirwpetcpecghrllkegkvhrcpnplcpakrfeairhfasrkamdigglgeklierllekglvkdvadlyrlrkedlvglermgeksacinllrgieeskkrglerllyalglpgvgevlarnlaarfgnmdrlleasleelleveevgeltarailetlkdpafrdlvrrlkeagvemeakekggealkgltfvitgelsrpreevkallrrlgakvtdsysrktsylvvgenpgsklekaralgvptlteeelyrllear tgkkaeeiv 4-2-5-8Mghhhhhhhhhhssghiegrasadgpylgileqpkgrgfrfryvcegps 13hgglpgasseknkksypqvkicnyvgpakvivqlvtngknihlhahslvgkhcedgictvtagpkdmvvgfanlgilhvtkkkvfetlearmteacirgynpgllvhpdlaylqaegggdrqlgdrekelirciaalqqtkemdlsvvrlmftaflpdstgsftrrlepvvsdaiydskapnasnlkivrmdrtagcvtggeeiyllcdkvqkddigirfyeeeenggvwegfgdfsptdvhrcifaivfktpkykdinitkpasvfvqlrrksdletsepkpflyypeikdkeevcirkrqkgssgtsgggsgggmilkilneiasigstkqkciaileknkdnellkrvyrltysrglqyyikkwpkpgiatqsfgmltltdmldfieftlatrkltgnaaieeltgyitdgkkddvevlrrvmmrdlecgasysiankvwpglipeqpqmlassydekginknikfpafaqlkadgarcfaevrgdelddvrllsragneylgldllkeelikmtaeargihpegvlidgelvyheqvkkepegldflfdaypenskakefaevaesrtasngiankslkgtisekeaqcmkfqvwdyvplveiyslpafrlkydvrfsklegmtsgydkvilienqvvnnldeakviykkyidqglegiilknidglwenarsknlykfkevidvdlkivgiyphrkdptkaggfilesecgkikvnagsglkdkagvksheldrtrimencinyyigkilececngwlksdgrtdyvklflpiairlredktka ntfedvfgdfhevtgl4-2-5-9 Mghhhhhhhhhhssghiegrasadgpylgileqpkgrgfrfryvcegps 14hgglpgasseknkksypqvkicnyvgpakvivqlvtngknihlhahslvgkhcedgictvtagpkdmvvgfanlgilhvtkkkvfetlearmteacirgynpgllvhpdlaylqaegggdrqlgdrekelirciaalqqtkemdlsvvrlmftaflpdstgsftrrlepvvsdaiydskapnasnlkivrmdrtagcvtggeeiyllcdkvqkddigirfyeeeenggvwegfgdfsptdvhrcifaivfktpkykdinitkpasvfvqlrrksdletsepkpflyypeikdkeevcirkrqkgssgtsgggsgggmrylelacilyqklekttmkliktrlvadflkkvpddhlefipylilgdvfpewderelgvgekllikavamatgidaneiensvkdtgdlgesialavkkrkqksffscipltikrvyqtivkvaettgegsgekkmkylanlfmdaepieakyiartvlgtmrtgvaegllrdaialafhvkvelveraymltsdfgfvakvaklegneglakvqvgigkpikpmlaqqaanikeallemggeaefeikydgarvqvhkdgdkiivysrrlenvtraipeivealkgsvkpnkaivegelvaigedgrplpfqyvlrrfrrkhniqemmkkiplelnlfdvlyvdgesmidvkfidrrkkleeiiepngkikvaenlitkkveeaeafykkalemgheglmakrldatyepgnrgkkwlkikptmenldlviigaewgegrrahllgsfilgaydpetgeflevgkvgsgftdedlveftkmlkpliikeegkrvwiepkivievtycleigkspkyksgfalrfpryvalrddkgpedadtieriaglyelgermkgkv 4-2-5-10mghhhhhhhhhhssghiegrasadgpylgileqpkgrgfrfryvcegps 15hgglpgasseknkksypqvkicnyvgpakvivqlvtngknihlhahslvgkhcedgictvtagpkdmvvgfanlgilhvtkkkvfetlearmteacirgynpgllvhpdlaylqaegggdrqlgdrekelirciaalqqtkemdlsvvrlmftaflpdstgsftrrlepvvsdaiydskapnasnlkivrmdrtagcvtggeeiyllcdkvqkddigirfyeeeenggvwegfgdfsptdvhrcifaivfktpkykdinitkpasvfvqlrrksdletsepkpflyypeikdkeevcirkrqkgssgtsgggsgggmrylelacilyqklekttmkliktrlvadflkkvpddhlefipylilgdvfpewderelgvgekllikavamatgidaneiensvkdtgdlgesialavkkrkqksffscipltikrvyqtivkvaettgegsgekkmkylanlfmdaepieakyiartvlgtmrtgvaegllrdaialafhvkvelveraymltsdfgfvakvaklegneglakvqvgigkpikpmlaqqaanikeallemggeaefeikydgarvqvhkdgdkiivysrrlenvtraipeivealkgsvkpnkaivegelvaigedgrplpfqyvlrrfrrkhniqemmkkiplelnlfdvlyvdgesmidvkfidrrkkleeiiepngkikvaenlitkkveeaeafykkalemgheglmakrldatyepgnrgkkwlkikptmenldlviigaewgegrrahllgsfilgaydpetgeflevgkvgsgftdedlveftkmlkpliikeegkrvwiepkivievtycleigkspkyksgf alrfpryvalrddkgp4-7-5-3 Mghhhhhhhhhhssghiegrassptsymspslpaldwqlpshsgpyelr 16ievqpkshhrahyetegsrgavkasagghpivqlhgylenepltlqlfigtaddrllrphafyqvhritgktvsttsheiilsntkvleipllpennmraiidcagilklrnsdielrkgetdigrkntrvrlvfrvhipqpngrtlslciasnlkivrmdrtagcvtggeeiyllcdkvqkddigirfyeeeenggvwegfgdfsptdvhrgfaivfktpkykdinitkpasvfvqlrrksdletsepkpflyypeikdkeevqrkrqkgssgtsgggsgggmtleearkrvnelrdliryhnyryyvladpeisdaeydrllrelkeleerfpelkspdsptlqvgarpleatfrpvrhptrmysldnafnldelkafeerieralgrkgpfaytvehkvdglsvnlyyeegvlvygatrgdgevgeevtqnlltiptiprrlkgvperlevrgevympieaflrineeleergerifknprnaaagslrqkdpritakrglratfyalglgleeveregvatqfallhwlkekgfpvehgyaravgaegveavyqdwlkkrralpfeadgvvvkldelalwrelgytaraprfaiaykfpaeeketrlldvvfqvgrtgrvtpvgilepvflegsevsrvtlhnesyieeldirigdwvlvhkaggvipevlrvlkerrtgeerpirwpetcpecghrllkegkvhrcpnplcpakrfeairhfasrkamdiqglgeklierllekglvkdvadlyrlrkedlvglermgeksacinllrgieeskkrglerllyalglpgvgevlarnlaarfgnmdrlleasleelleveevgeltarailetlkdpafrdlvrrlkeagvemeakekggealkgltfvitgelsrpreevkallrrlgakvtdsysrktsylvvgenpgsklekaralgvptlteeelyrlleartgkkaeelv 4-7-5-8Mghhhhhhhhhhssghiegrassptsymspslpaldwqlpshsgpyelr 17ievqpkshhrahyetegsrgavkasagghpivqlhgylenepltlqlfigtaddrllrphafyqvhritgktvsttsheiilsntkvleipllpennmraiidcagilklrnsdielrkgetdigrkntrvrlvfrvhipqpngrtlslciasnlkivrmdrtagcvtggeeiyllcdkvqkddigirfyeeeenggvwegfgdfsptdvhrgfaivfktpkykdinitkpasvfvqlrrksdletsepkpflyypeikdkeevqrkrqkgssgtsgggsgggmilkilneiasigstkqkciaileknkdnellkrvyrltysrglqyyikkwpkpgiatqsfgmltltdmldfieftlatrkltgnaaieeltgyitdgkkddvevlrrvmmrdlecgasysiankvwpglipeqpqmlassydekginknikfpafaqlkadgarcfaevrgdelddvrllsragneylgldllkeelikmtaearqihpegvlidgelvyheqvkkepegldflfdaypenskakefaevaesrtasngiankslkgtisekeaqcmkfqvwdyvplveiyslpafrlkydvrfsklegmtsgydkvilienqvvnnldeakviykkyidgglegiilknidglwenarsknlykfkevidvdlkivgiyphrkdptkaggfilesecgkikvnagsglkdkagvksheldrtrimencinyyigkilececngwlksdgrtdyvklflpiairlredktkantfedvfgdfhevtgl 4-7-5-9Mghhhhhhhhhhssghiegrassptsymspslpaldwqlpshsgpyelr 18ievqpkshhrahyetegsrgavkasagghpivqlhgylenepltlqlfigtaddrllrphafyqvhritgktvsttsheiilsntkvleipllpennmraiidcagilklrnsdielrkgetdigrkntrvrlvfrvhipqpngrtlslciasnlkivrmdrtagcvtggeeiyllcdkvqkddigirfyeeeenggvwegfgdfsptdvhrgfaivfktpkykdinitkpasvfvqlrrksdletsepkpflyypeikdkeevqrkrqkgssgtsgggsgggmrylelacilyqklekttmkliktrlvadflkkvpddhlefipylilgdvfpewderelgvgekllikavamatgidaneiensvkdtgdlgesialavkkrkqksffsqpltikrvyqtivkvaettgegsgekkmkylanlfmdaepieakyiartvlgtmrtgvaegllrdaialafhvkvelveraymltsdfgfvakvaklegneglakvqvgigkpikpmlaqqaanikeallemggeaefeikydgarvqvhkdgdkiivysrrlenvtraipeivealkqsvkpnkaivegelvaigedgrplpfqyvlrrfrrkhniqemmkkiplelnlfdvlyvdgesmidvkfidrrkkleeiiepngkikvaenlitkkveeaeafykkalemgheglmakrldatyepgnrgkkwlkikptmenldlviigaewgegrrahllgsfilgaydpetgeflevgkvgsgftdedlveftkmlkpliikeegkrvwiepkivievtycleigkspkyksgfalrfpryvalrddkgpedadtieriacilye lgermkgkv 4-7-5-10Mghhhhhhhhhhssghiegrassptsymspslpaldwqlpshsgpyelr 19ievqpkshhrahyetegsrgavkasagghpivqlhgylenepltlqlfigtaddrllrphafyqvhritgktvsttsheiilsntkvleipllpennmraiidcagilklrnsdielrkgetdigrkntrvrlvfrvhipqpngrtlslciasnlkivrmdrtagcvtggeeiyllcdkvqkddigirfyeeeenggvwegfgdfsptdvhrgfaivfktpkykdinitkpasvfvqlrrksdletsepkpflyypeikdkeevqrkrqkgssgtsgggsgggmrylelacilyqklekttmkliktrlvadflkkvpddhlefipylilgdvfpewderelgvgekllikavamatgidaneiensvkdtgdlgesialavkkrkqksffsqpltikrvyqtivkvaettgegsgekkmkylanlfmdaepieakyiartvlgtmrtgvaegllrdaialafhvkvelveraymltsdfgfvakvaklegneglakvqvgigkpikpmlaqqaanikeallemggeaefeikydgarvqvhkdgdkiivysrrlenvtraipeivealkqsvkpnkaivegelvaigedgrplpfqyvlrrfrrkhniqemmkkiplelnlfdvlyvdgesmidvkfidrrkkleeiiepngkikvaenlitkkveeaeafykkalemgheglmakrldatyepgnrgkkwlkikptmenldlviigaewgegrrahllgsfilgaydpetgeflevgkvgsgftdedlveftkmlkpliikeegkrvwiepkivievtygeigkspkyksgfalrfpryvalrddkgp

The above-indicated fusion protein sequences provide examples ofblunt-end ligases including DNA-ligase portions. Further fusion proteinsbelieved to be useful as blunt-end ligases, and believed to be useful asthermostable nucleic acid blunt-end ligases, include further fusionproteins comprising variants of the fusion protein sequences describedherein by SEQ ID NO:. In embodiments, such further fusion proteinsbelieved to be useful as blunt-end ligases, and believed to be useful asthermostable nucleic acid blunt-end ligases, are homologous variantsvariants of the fusion protein sequences described herein by SEQ ID NO:,e.g., homologous variants having greater than 90%, or greater than 95%,or greater than 99% sequence homology or sequence identity to a fusionprotein sequence described herein by SEQ ID NO:.

As discussed above, a second amino acid sequence having sequencehomology to a first amino acid sequence may differ from the first aminoacid by only conservative substitutions; that is, a residue in thesecond amino acid sequence is either a) identical to the correspondingresidue in the first amino acid sequence; b) a member of the same groupof amino acids as the corresponding residue in the first amino acidsequence, where the group is based on common side-chain properties asdisclosed above: or c) an exemplary or preferred substitution (asidentified above in Table 1C above).

For example, suitable fusion proteins include fusion proteins havinggreater than 90%, or greater than 95% or greater than 99%, sequencehomology or sequence identity to a fusion protein having a composition4-DNA binding protein-5-DNA ligase (written in the N-terminal toC-terminal orientation), where “DNA binding protein” indicates the aminoacid sequence of a DNA binding protein and “DNA ligase” indicates theamino acid sequence of a DNA ligase included in the fusion protein. Asdiscussed above, examples of DNA binding proteins include SEQ ID NO: 2and SEQ ID NO: 7; examples of DNA ligases include SEQ ID NO: 3, SEQ IDNO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.

Example 3

Further fusion proteins believed to be useful as blunt-end ligases, andbelieved to be useful as thermostable nucleic acid blunt-end ligases,include fusion proteins as described herein, where the DNA-bindingprotein is replaced by a RNA-binding protein, and where the ligasecomprises an RNA-ligase protein in fusion proteins having a composition4-RNA binding protein-5-RNA ligase (written in the N-terminal toC-terminal orientation), where “RNA binding protein” indicates the aminoacid sequence of a RNA binding protein and “RNA ligase” indicates theamino acid sequence of a RNA ligase included in the fusion protein. Inembodiments, the RNA ligase may be a thermostable RNA ligase. Thus, afusion protein as disclosed herein having RNA ligase activity, andbelieved to have thermostable RNA ligase activity, comprises a linearpolypeptide having a composition including the covalently linked aminoacid sequences (in the following N-terminal to C-terminal order) SEQ IDNO: 4, RNA binding protein, SEQ ID NO: 5, RNA ligase.

In addition, suitable RNA ligase fusion proteins include RNA ligasefusion proteins having greater than 90%, or greater than 95% or greaterthan 99%, sequence homology to a fusion protein having a composition4-RNA binding protein-5-RNA ligase. As discussed above, a second aminoacid sequence having sequence homology to a first amino acid sequencemay differ from the first amino acid by only conservative substitutions;that is, a residue in the second amino acid sequence is either a)identical to the corresponding residue in the first amino acid sequence;b) a member of the same group of amino acids as the correspondingresidue in the first amino acid sequence, where the group is based oncommon side-chain properties as disclosed above: or c) an exemplary orpreferred substitution (as identified above in Table 1C above).

Example 4

Proteins having features as disclosed herein include proteins that arehomologous to any of the fusion proteins disclosed herein. Such proteinsare believed to be suitable for use as nucleic acid ligases, includingas thermostable nucleic acid ligases. For example, such proteins arebelieved to be suitable for use as blunt-end DNA ligases, including asthermostable blunt-end DNA ligases. Such proteins are further believedto be suitable for use as blunt-end RNA ligases, including asthermostable blunt-end RNA ligases.

Proteins that are homologous to any of the fusion proteins disclosedherein may be provided by providing variant proteins having greater than90%, or greater than 95% or greater than 99%, sequence homology to afusion protein disclosed herein. For example, particular proteins thatare homologous to any of the fusion proteins disclosed herein may beprovided by providing variant proteins having greater than 90%, orgreater than 95% or greater than 99%, sequence homology or sequenceidentity to SEQ ID NO: 1, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15,SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19 (i.e.,having greater than 90%, or greater than 95% or greater than 99%,sequence homology or sequence identity to a fusion protein of Table 4).

Fusion proteins as disclosed herein include a composition having thegeneral form of a linear molecule having a histidine-rich leadersequence covalently linked to a nucleic acid binding protein sequencewhich is covalently linked to a flexible glycine-rich sequence which iscovalently linked to a nucleic acid ligase sequence. In embodiments,such fusion proteins may be proteins including one or more amino acidsubstitutions, insertions, deletions, or additions as compared to afusion protein sequence provided as a sequence listing herein. Inembodiments, such fusion proteins may be proteins including one or moreamino acid substitutions, insertions, deletions, or additions ascompared to a histidine-rich leader sequence, a nucleic acid bindingprotein sequence, a flexible glycine-rich sequence, or a nucleic acidligase sequence provided as a sequence listing herein.

For example, homologous fusion proteins having features disclosed hereinmay include histidine-rich leader sequences different than, whilehomologous to, SEQ ID NO: 4; e.g., having histidine-rich leadersequences having greater than 90%, or greater than 95% or greater than99%, sequence homology or sequence identity to SEQ ID NO: 4. It will beunderstood that such homologous histidine-rich leader sequences mayinclude the same number, or may include a greater number, or may includea smaller number, of amino acid residues than SEQ ID NO: 4.

For example, homologous fusion proteins having features disclosed hereinmay include flexible glycine-rich sequences different than, whilehomologous to, SEQ ID NO: 5; e.g., having flexible glycine-richsequences having greater than 90%, or greater than 95% or greater than99%, sequence homology or sequence identity to SEQ ID NO: 5. It will beunderstood that such homologous flexible glycine-rich sequences mayinclude the same number, or may include a greater number, or may includea smaller number, of amino acid residues than SEQ ID NO: 5.

For example, homologous fusion proteins include fusion proteins havingcompositions homologous to any of the proteins described herein as4-2-5-3, 4-2-5-8, 4-2-5-9, and 4-2-5-10, where the sequence “2” isreplaced by a sequence having greater than 90%, or greater than 95% orgreater than 99%, sequence homology or sequence identity to SEQ ID NO:2; or where the sequence “3”, or “8”, or “9”, or “10” is replaced by asequence having greater than 90%, or greater than 95% or greater than99%, sequence homology or sequence identity to SEQ ID NO: 3, SEQ ID NO:8, SEQ ID NO: 9, or SEQ ID NO: 10. In embodiments, both the sequence “2”is replaced by a homologous sequence and the sequence “3”, or “8”, or“9”, or “10” is replaced by a homologous sequence.

In addition, homologous fusion proteins include fusion proteins havingcompositions homologous to any of the proteins described herein as4-7-5-3, 4-7-5-8, 4-7-5-9, and 4-7-5-10, where the sequence “7” isreplaced by a sequence having greater than 90%, or greater than 95% orgreater than 99%, sequence homology or sequence identity to SEQ ID NO:7; or where the sequence “3”, or “8”, or “9”, or “10” is replaced by asequence having greater than 90%, or greater than 95% or greater than99%, sequence homology or sequence identity to SEQ ID NO: 3, SEQ ID NO:8, SEQ ID NO: 9, or SEQ ID NO: 10. In embodiments, both the sequence “7”is replaced by a homologous sequence and the sequence “3”, or “8”, or“9”, or “10” is replaced by a homologous sequence.

While the above is a complete description of the preferred embodiment asdescribed herein, it is possible to use various alternatives,modifications and equivalents. Therefore, the scope of the presentinvention should be determined not with reference to the abovedescription but should, instead, be determined with reference to theappended claims, along with their full scope of equivalents. Anyfeature, whether preferred or not, may be combined with any otherfeature, whether preferred or not. The appended claims are not to beinterpreted as including means-plus-function limitations, unless such alimitation is explicitly recited in a given claim using the phrase“means for.” It should be understood that as used in the descriptionherein and throughout the claims that follow, the meaning of “a,” “an,”and “the” includes plural reference unless the context clearly dictatesotherwise. Also, as used in the description herein and throughout theclaims that follow, the meaning of “in” includes “in” and “on” unlessthe context clearly dictates otherwise. Finally, as used in thedescription herein and throughout the claims that follow, the meaningsof “and” and “or” include both the conjunctive and disjunctive and maybe used interchangeably unless the context expressly dictates otherwise.Thus, in contexts where the terms “and” or “or” are used, usage of suchconjunctions do not exclude an “and/or” meaning unless the contextexpressly dictates otherwise.

This document contains material subject to copyright protection. Thecopyright owner (Applicant herein) has no objection to the facsimilereproduction by anyone of the patent document or the patent disclosure,as they appear in the US Patent and Trademark Office patent file orrecords, but otherwise reserves all copyright rights whatsoever. Thefollowing notice shall apply: Copyright 2013-2014 Theranos, Inc.

1. A thermostable blunt-end DNA ligase comprising a fusion proteincomprising, in N-terminal to C-terminal order, a leader sequencecomprising the amino acid sequence SEQ ID NO: 4, a DNA binding protein,a glycine-rich amino acid sequence comprising SEQ ID NO: 5, and a DNAligase, wherein said thermostable blunt-end DNA ligase is suitable foruse in a blunt-ended DNA ligation reaction performed at about 60° C. orhigher.
 2. The thermostable blunt-end DNA ligase of claim 1, comprisinga T4 DNA ligase with an N-terminal p50 fusion.
 3. The thermostableblunt-end DNA ligase of claim 1, comprising an amino acid sequenceselected from the group of amino acid sequences consisting of SEQ ID NO:1, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ IDNO: 17, SEQ ID NO: 18, and SEQ ID NO:
 19. 4. A method of ligatingblunt-end DNA at elevated temperature, comprising ligating blunt-end DNAusing a thermostable blunt-end DNA ligase suitable for use in ablunt-ended DNA ligation reaction performed at about 60° C. or higher,wherein said thermostable blunt-end DNA ligase comprises a thermostableblunt-end DNA ligase of claim
 1. 5. An article of manufacture,comprising a thermostable blunt-end DNA ligase suitable for use in ablunt-ended DNA ligation reaction performed at about 60° C. or higherand a container, wherein said thermostable blunt-end DNA ligasecomprises a thermostable blunt-end DNA ligase of claim
 1. 6. The articleof manufacture of claim 5, further comprising a buffer.
 7. A device foranalyzing a sample containing DNA, comprising a thermostable blunt-endDNA ligase suitable for use in a blunt-ended DNA ligation reactionperformed at about 60° C. or higher, wherein said thermostable blunt-endDNA ligase comprises a thermostable blunt-end DNA ligase of claim
 1. 8.The method of claim 4, wherein said thermostable blunt-end DNA ligase issuitable for use in a blunt-ended DNA ligation reaction performed atabout 75° C., and said ligating is performed at about 75° C.
 9. Thethermostable blunt-end DNA ligase of claim 1, wherein said thermostableblunt-end DNA ligase is capable of making concatamers upon multipleligation events in a blunt-ended DNA ligation reaction performed atabout 60° C. or higher.
 10. The thermostable blunt-end DNA ligase ofclaim 1, wherein said thermostable blunt-end DNA ligase is suitable foruse in a nucleic acid amplification scheme which operates at a uniformtemperature of about 60° C. or higher. 11-15. (canceled)
 16. Thethermostable blunt-end DNA ligase of claim 1, wherein said fusionprotein comprises a component selected from a peptide linker, anN-terminal addition, a C-terminal addition, a tag peptide, a D-aminoacid, and a peptide mimetic.
 17. The thermostable blunt-end DNA ligaseof claim 1, wherein said fusion protein comprises a component selectedfrom a sugar and a polymer. 18-21. (canceled)
 22. A method of ligatingblunt-end nucleic acids at an elevated temperature, comprising using athermostable blunt-end nucleic acid ligase of claim 3 at a temperatureof about 60° C. or higher.
 23. A device for analyzing a samplecontaining nucleic acids, comprising a thermostable blunt-end nucleicacid ligase of claim
 1. 24. The device of claim 23, wherein saidthermostable blunt-end ligase comprises an amino acid sequence selectedfrom SEQ ID NO: 1, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ IDNO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO:
 19. 25. A fusionprotein comprising the amino acid sequence SEQ ID NO: 20 and a nucleicacid ligase.
 26. The fusion protein of claim 25, wherein the nucleicacid ligase is a DNA ligase.
 27. The fusion protein of claim 25, whereinthe nucleic acid ligase is a RNA ligase.
 28. The fusion protein of claim25, wherein the nucleic acid ligase is a thermostable DNA ligase. 29.The fusion protein of claim 25, wherein the nucleic acid ligase is athermostable RNA ligase.